Diabetes Mellitus is a serious disease afflicting over 100 million people worldwide. In the United States, there are more than 12 million diabetics, with 600,000 new cases diagnosed each year. Insulin-dependent, or Type I diabetics, require daily injections of insulin to prevent them from lapsing into coma.
With the discovery of insulin in 1928, it was thought that diabetes had been cured. Unfortunately, despite insulin therapy, the major complications of the disease caused by high blood sugar levels persist. Each year, diabetes accounts for 40,000 limb amputations and 5,000 new cases of blindness in the United States. Among teenagers, diabetes is the leading cause of kidney failure. Data from the National Institutes of Health show that the rate of heart disease and stroke is twice as great in diabetics than in the general population.
The diabetic patient faces a 30% reduced lifespan. Multiple insulin injections given periodically throughout the day cannot duplicate the precise feedback of insulin secretion from the pancreas. The only current method of achieving minute-to-minute glucose control is by pancreas transplantation.
While whole organ pancreas transplantation represents a significant advance in diabetes therapy, the operation is technically difficult, of limited success and use because of the problems of rejection, and it still presents a significant risk to the patient. An attractive alternative would be to extract the insulin-producing cells (islets) from a donor pancreas and to inject these cells into the diabetic patient, thus effecting a cure. The use of such cells, however, would still run the risk of rejection by the host.
Microencapsulation of islets by an alginate-PLL-alginate membrane (i.e., an alginate-poly-L-lysine-alginate membrane) is a potential method for prevention of rejection by the host's immune system. By this technique, researchers are able to encapsulate living islets in a protective membrane that allows insulin to be secreted, yet prevents antibodies from reaching the islets, causing rejection of the cells. This membrane (or microcapsule) protects the islet from rejection and allows insulin to be secreted through its "pores" to maintain the diabetic in normal glucose control.
Successful transplants of microencapsulated islets have not been clinically feasible to date due to fundamental problems of transplant rejection and/or a fibrotic reaction to the microcapsule membrane. Lim and Sun, 1980, reported the first successful implantation of microencapsulated islets and described normalization of blood sugar in diabetic rats. However, for microencapsulated islets to be clinically useful and applicable in humans, it is important that the immunoprotective membrane be biocompatible, allow adequate diffusion for the encapsulated cells to respond appropriately to a stimulatory signal, provide the encapsulated cells with necessary nutrients, and be retrievable. Retrievability is desirable for a variety of reasons, e.g., so that accumulation of the implanted materials can be avoided, so that encapsulated cells can be removed from the recipient when no longer needed or desired (e.g., when the product(s) of the encapsulated cells are no longer needed, if the encapsulated cells fail to perform as desired, etc.), so that encapsulated cells can be removed if/when they become non-viable, and the like. Currently there are no reports of successful reversal of diabetes in humans by transplantation of encapsulated islets.
Biocompatibility of encapsulated islets remains a fundamental problem. The term "biocompatible" is used herein in its broad sense, and relates to the ability of the material to result in long-term in vivo function of transplanted biological material, as well as its ability to avoid a foreign body, fibrotic response. A major problem with microencapsulation technology has been the occurrence of fibrous overgrowth of the epicapsular surface, resulting in cell death and early graft failure. Despite extensive studies, the pathological basis of this phenomenon in alginate based capsules remains poorly understood. However, several factors have recently been identified as being involved in graft failure, e.g., the guluronic acid/mannuronic acid content of the alginate employed, imperfections in the microcapsule membrane (allowing exposure of poly-L-lysine to the in vivo environment), failure of the microcapsule membrane to completely cover the cells being encapsulated (thereby allowing exposure of the cells to the in vivo environment), and the like.
Alginate is a polysaccharide isolated from marine brown algae including Laminaria hyperborea, Laminaria digitata, Ascophyllum nodosum and Macrocystis pyrifera. Alginate forms ionically crosslinked gels with most di- and multivalent cations. Calcium cations are most widely used, and give rise to a three-dimensional network in the form of an ionically crosslinked gel by inter-chain binding between G-blocks. (Skjak-Br.ae butted.k, 1988).
It has recently been demonstrated that the mannuronic acid residues are the active cytokine inducers in alginate, and since these cytokines (IL-1 and TNF) are known to be potent stimulators of fibroblast proliferation (Otterlei et al., 1991), it was deduced that alginate capsules high in mannuronic acid content (M-content) were responsible in part for the fibrotic response reported in the past (Soon-Shiong et al. 1991). More significantly, it has been found that this reaction could be ameliorated by increasing the guluronic acid content (G-content) of the alginate capsule since guluronic acid appears not to be immunostimulating. Furthermore, it has been demonstrated that cyclosporin A resulted in a dose dependent inhibition of mannuronic acid-induced TNF and IL-1 stimulation of human monocytes in vitro. Based on this information, it has been hypothesized that the fibrotic reaction of the microcapsule could be ameliorated in part by an alginate formulation high in guluronic acid content, as well as a subtherapeutic course of cyclosporin A to inhibit cytokine stimulation. By this method, diabetes in the spontaneous diabetic dog model has been successfully reversed by transplantation of donor islets encapsulated in high G-content alginate (Soon-Shiong et al. 1991).
Polyethylene glycols (PEG; also referred to as polyethylene oxide, PEO) have been investigated extensively in recent years for use as biocompatible, protein repulsive, noninflammatory, and nonimmunogenic modifiers for drugs, proteins, enzymes, and surfaces of implanted materials. The basis for these extraordinary characteristics has been attributed to the flexibility of the polymer backbone, and the volume exclusion effect of this polymer in solution or when immobilized at a surface. The solubility of PEGs in water, as well as a number of common organic solvents, facilitates modification by a variety of chemical reactions. A recent review (Harris, 1985) describes the synthesis of numerous derivatives of PEG and the immobilization thereof to surfaces, proteins, and drugs.
PEG bound to bovine serum albumin has shown reduced immunogenicity and increased circulation times in a rabbit (Abuchowski et al., 1977). Drugs such as penicillin, aspirin, amphetamine, quinidine, procaine, and atropine have been attached to PEG in order to increase their duration of activity as a result of slow release (Weiner et al., 1974; Zalipsky et al., 1983). PEG covalently bound to poly-L-lysine (PLL) has been used to enhance the biocompatibility of alginate-PLL microcapsules used for the encapsulation of cells. PEG has been covalently bound to polysaccharides such as dextran (Pitha et al., 1979, Duval et al., 1991), chitosan (Harris et al., 1984) and alginates (Desai et al., 1991). These modifications confer organic solubility to the polysaccharides.
Surfaces modified with PEG were found to be extremely nonthrombogenic (Desai and Hubbell, 1991a; Nagoaka and Nakao, 1990), resistant to fibrous overgrowth in vivo (Desai and Hubbell, 1992a) and resistant to bacterial adhesion (Desai and Hubbell, 1992b). Solutions containing PEG have also been found to enhance the preservation of organs for transplantation (Collins et al.; Zheng et al., 1991). The basis of the preservation activity is not clearly understood but has been attributed to adhesion of PEG to cell surface molecules with a resultant change in the presentation of antigen so as to alter the nature of the immune response.
Crosslinked PEG gels have been prepared and utilized for immobilization of enzymes and microbial cells. Fukui and Tanaka (1976) and Fukui et al., (1987) have prepared polymerizable derivatives of PEG (such as the dimethacrylate) and photocrosslinked them with UV light in the presence of a suitable initiator to form a covalently crosslinked gel. Kumakura and Kaetsu (1983) have reported the polymerization and crosslinking of diacrylate derivatives of PEG by gamma radiation for the purpose of immobilizing microbial cells. Due to the mild nature of the photopolymerization, i.e., absence of heating, without shifting pH to extreme values, and without the use of toxic chemicals, the Fukui and Tanaka (1976) publication suggests that this technique is desirable for the entrapment not only of enzymes, but also for cells and organelles.
Dupuy et al. (1988) have recently described a photopolymerization process for the entrapment of agarose embedded pancreatic islets in microspheres of crosslinked acrylamide. However, this reference describes entrapment of individual microspheres, but does not describe further entrapment of these already entrapped cells. Visible light was used as the initiating radiation in the presence of a photochemical sensitizer (vitamin B2, i.e., riboflavin), and a cocatalyst (N-N-N'N'-tetramethylethylene diamine). A high pressure mercury lamp was used as the source of visible radiation and the islets were demonstrated to maintain a good viability in vitro following the polymerization step.
Visible radiation between wavelengths of 400-700 nm have been determined to be nontoxic to living cells (Karu, 1990; Dupuy et al., 1988). A recent review (Eaton, 1986) describes a variety of dyes and cocatalysts that may be used as polymerization initiators in the presence of appropriate visible radiation.
In recent years considerable interest has been expressed in the use of lasers for polymerization processes (Wu, 1990). These polymerizations are extremely fast and may be completed in milliseconds (Decker and Moussa, 1989; Hoyle et al., 1989; Eaton, 1986). The use of coherent radiation often results in the polymerization being innocuous to living cells. This arises from the use of wavelength specific chromophores as the polymerization initiators, and these chromophores are typically the only species in the polymer/cell suspension that absorb the incident radiation.